DHAP-dependent aldolases catalyze the C-C bond condensation of DHAP and an aldehyde. The products of this reaction show high therapeutic value as drugs or pharmaceutical intermediates. While these aldolases have potential for industrial applications, the enzymes described to date have stability problems and are limited by their substrate specificities. The application of microbial enzymes for organic synthetic reactions can offer high substrate specificity, complete substrate conversion under mild reaction conditions, and less chemical waste generation. Thermostable enzymes in general offer all these features as well as enhanced stability. ThermoGen proposes to develop a set of stable DHAP-dependent aldolases and an efficient, scaleable technology for use in organic synthesis reactions. In Phase I we will show feasibility by identifying a set of four enzymes with different stereoselectivities made up of either new aldolases or mutagenized aldolases with broader substrate specificities and stability. We will also identify key reaction parameters to test synthetic utility and demonstrate their ability to catalyze model synthetic reactions. In Phase II we will develop methods for optimal synthesis conditions, enzyme production, and expand the substrate range. In Phase III we will commercialize the enzymes and technology for use by synthetic organic chemists and pharmaceutical manufacturers. PROPOSED COMMERCIAL APPLICATIONS: ThermoGen proposes to develop novel DHAP-dependent aldolases for use as synthetic tools in stereoselective organic synthetic reactions. Aldolases will help syntheses or pharmaceutical drugs or intermediates efficiently and economically and reduce the chemical waste generation.